RU2491059C2 - Product of iron source in form of particles and method of their obtaining - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to iron-enriched food product, which contains iron source in form of solid particles, where particles include core, containing iron alginate, and external layer, which contains calcium alginate, where particles are obtained by method which includes the following stages: (i) formation of core, which contains iron alginate, by contact of bioavailable water-soluble salt of iron and one water-soluble alginate salt, (ii) contact of core with water solution of calcium salt, in concentration, which constitutes from 0.025 M to concentration of lower than solution saturation point, and (iii) separation of obtained solid product. Iron-enriched food product is applied for prevention and treatment of iron deficiency conditions of people.

EFFECT: solid particles are applicable for enrichment of food products with iron and is characterised by improved load ability, as well as possesses good stability under standard storage and application conditions.

11 cl, 9 dwg, 13 tbl, 16 ex

Description

This application claims the priority of provisional patent application US No. 61/114261, filed November 13, 2008 and European patent application EP08166052.4, which are incorporated herein by reference.

The technical field of the invention

The present invention relates to iron-fortified foods and their use to prevent iron deficiency or to reduce iron deficiency in the human body.

BACKGROUND OF THE INVENTION

Iron deficiency is one of the most common diseases caused by nutritional deficiencies found in most developing countries, as well as a major disease caused by nutritional deficiencies in industrialized countries. Enriching certain foods with iron is one way to prevent iron deficiency. However, in order for fortified foods to be effective in reducing iron deficiency, the added iron must be sufficiently biologically available.

A suitable iron fortifying agent must satisfy a number of requirements. Firstly, it must be harmless to the human body. In addition, it must be insoluble in water in a neutral or moderately acidic environment, which is a decisive factor for long-term storage. It should also have a high absorption capacity in the human body, that is, good bioavailability (which means good solubility in the gastrointestinal tract). In addition, it must have good stability and a determinable chemical composition, and be produced in a reproducible manner, that is, it must have guaranteed consistent and controlled properties.

The bioavailability of iron depends on its chemical form, on which its solubility depends, as well as on the presence of food components, either contributing to or preventing its absorption. Bioavailability is closely related to sensory changes. Water-readily soluble sources of iron, such as iron sulfate, iron gluconate, iron lactate and the double salt of iron citrate and ammonium citrate, have a relatively high bioavailability, however, they have drawbacks associated with the discoloration and taste change of the products enriched in them when they enter reaction with other components of food. Iron fumarate, iron succinate, and iron sugar are slowly soluble in water, but readily soluble in dilute acids such as gastric juice. Although it may seem that they have advantages over iron sulfate in terms of their effect on fat oxidation and discoloration, their interaction with food can reduce iron absorption. The absence of any interaction with food during storage of poorly soluble sources of iron, such as iron pyrophosphate, iron orthophosphate and elemental iron, makes them attractive enrichment substances from a commercial point of view, and now many products from crushed grain contain such forms of iron. However, the bioavailability of these iron sources is always low.

Thus, conventional commercially available iron compounds turn out to be either sufficiently soluble in water to cause technical problems, or so difficult to dissolve that their absorption capacity in the human body is low.

Attempts have been made to create a stable bioavailable source of iron for fortifying food by encapsulating it with an inert compound to protect it from oxidation and minimize its organoleptic effect. However, the encapsulation of certain iron salts with hydrogenated soybean oil, mono- and diglycerides, or ethyl cellulose, although it provides some protection for the iron salt, is not found to be suitable for enriching certain foods (see RF Hurrell et al. Iron fortification of infant cereals: a proposal for the use of ferrous fumarate or ferrous succinate ", Am. J. Clin. Nutr. 1989, vol. 49, pp. 1274).

EP-A-1694312 discloses monohydrated iron sulfate particles coated with a layer of sodium alginate. To obtain particles, a solution of sodium alginate is sprayed onto the surface of the solid particles of iron sulfate with stirring. A thin layer of alginate solution is deposited on the particles, which contributes to the formation of a film of iron alginate, covering the unmodified core of iron sulfate. Upon contact with water, the particles slowly dissolve, releasing the core from iron sulfate into the medium. Accordingly, these particles can be included in dehydrated foods, such as wheat flour and other cereals, but they are not suitable for fortifying foods containing water, such as yoghurts or juices.

In addition, chelating agents have been shown to be effective in increasing the bioavailability of iron and iron salts. The combination of iron and sodium salt EDTA is considered a promising iron fortifier. The binding of EDTA to iron is facilitated by the acidic environment of the stomach, but in a more alkaline environment of the duodenum, iron is partially exchanged for other metals. Based on some studies involving animals and humans, it has been suggested that iron dissociates from the complex with EDTA in the intestinal lumen before it is absorbed, so that it can be transported using a highly regulated transcellular DMT-1 metabolic pathway. It has also been reported that a combination of iron and EDTA protects iron from exposure to other iron absorption inhibitors, such as phytates or polyphenols. Its potential as an iron fortifier has been confirmed in five extensive food fortification trials conducted in developing countries (L. Zhu et al. “Iron dissociates from the NaFeEDTA complex prior to or during intestinal absorption in rats”, J. Agric. Food Chem. 2006, Vol. 54, 7929-34). Some short-chain organic acids, such as tartaric acid, malic acid, succinic acid, and fumaric acid, show up to a 40-fold increase in the bioavailability of iron compounds in in vitro assays using CACO-2 cells (S. Salovaara et al., " Organic acids influence iron uptake in the human epithelial cell line Caco-2 ", J. Agric. Food Chem. 2002, Vol.50, p. 6233). The proposed mechanism of action is similar to that for EDTA. The chelating agent binds the cation of iron, Fe (II) or Fe (III), and saves it from precipitation due to alkaline pH or any other compound that traps and precipitates iron. Iron can then diffuse to enterocytes, where it can be absorbed.

Although various sources of iron for food fortification are already known, there is still a need for an iron fortifying food product that can be used with water-containing food products and which has good mechanical strength, while ensuring high bioavailability in the human body and good stability fortified foods during storage.

SUMMARY OF THE INVENTION

The inventors found a product that is a bioavailable source of iron with improved physical properties that does not dissolve in water or in slightly acidic environments, when added to food products, the iron-fortified foods do not undergo color changes and do not become rancid, even if they are high water. At the same time, the new iron source product is sufficiently soluble at pH of the stomach (which in an empty stomach can even correspond to 1) to ensure good bioavailability. This iron source product is easy to handle, has good mechanical strength and can be systematically reproduced.

Thus, one aspect of the present invention relates to an iron-fortified food product comprising a particulate solid source iron product, wherein the particles include a core containing iron alginate and an outer layer containing calcium alginate.

Another aspect of the present invention relates to the use of the above iron-fortified food product to prevent iron deficiency or to reduce iron deficiency in the human body.

DETAILED DESCRIPTION OF THE INVENTION

The terms “bioavailability” and “bioavailable” refer to the extent to which a nutrient or trace element can be absorbed and used by the body. For the purposes of the invention, the iron salts used to produce the particulate iron source product used in the iron-fortified food product of the present invention should have good bioavailability, which means good solubility in the gastrointestinal tract. Accordingly, the terms “bioavailable iron salt”, “bioavailable water-soluble iron salt” and “water-soluble iron salt” as used herein mean any iron salt that is freely soluble in water, such as iron sulfate, iron gluconate, iron lactate and double citrate iron and ammonium citrate, as well as any salt of iron, which is slowly soluble in water but easily soluble in dilute acids, such as iron fumarate, iron succinate and iron sugar.

Water-soluble alginate salts, such as sodium, potassium, magnesium or ammonium alginate, are naturally occurring linear polysaccharides from marine brown algae, consisting of two types of monomers, residues of beta-D-mannuronic acid (M) and alpha-L-guluronic acid (G) arranged in an irregular manner and blocks along the chain. A biopolymer carrying carboxyl groups is capable of forming complexes with polyvalent metal ions.

When a water-soluble iron salt comes into contact with a water-soluble alginate salt, crosslinking and gelation of the carboxylic alginate groups occurs as a result of reaction with iron cations such as Fe ²⁺ or Fe ³⁺ .

It was found that when a core containing iron alginate is in contact with an aqueous solution of calcium salt, a particle is formed (formed by a core coated with an outer layer containing calcium alginate) as a result of the reaction of the alginate salt with calcium cations. This outer layer, being insoluble in water or in weak acids, eliminates the contact of iron with the environment while increasing the mechanical strength of the particles. This particulate iron source product is suitable for enriching water-containing foods.

In general, a food product that will be enriched with the aforementioned particulate iron source product is a food or drink, in particular a food or drink that is sensitive to oxidation, unpleasant taste, or discoloration in the presence of free iron. In particular, the iron source product can be used to enrich water-containing foods, such as yogurt, milk, broths, sauces, juices and other drinks, as well as commonly fortified foods, such as bulk products, wheat flour and other grains, as well as food products derived from them, such as bread, pasta and pastries. Additional examples of food products that can be enriched according to the invention are meat emulsions, such as sausages.

When forming the core, if the amount of iron salt is higher than necessary for the reaction with all monomers of the alginate salt, the iron salt will be captured by the gel formed. On the contrary, if the amount of water-soluble alginate salt is higher than necessary for the reaction with all available polyvalent cations, the water-soluble alginate salt will also be part of the gel formed. Accordingly, in an embodiment of the first aspect of the present invention, the core further comprises at least one bioavailable iron salt. In another embodiment of the same aspect of the invention, the core further comprises at least one water-soluble alginate salt, such as sodium, potassium, magnesium or ammonium alginate. Preferably, the water soluble alginate salt is sodium alginate.

Water soluble iron salts improve the bioavailability of iron and are therefore preferred. Thus, preferably, the bioavailable iron salt is a water-soluble iron salt such as iron sulfate, iron gluconate, iron lactate and a double salt of iron citrate and ammonium citrate, or slowly soluble in water but readily soluble in dilute acids such as iron fumarate, iron succinate and iron sugar. Preferably, the bioavailable iron salt is selected from iron sulfate and iron sugar. More preferably, the bioavailable iron salt is iron sugar.

In addition, as mentioned above, chelating agents have been shown to be effective in increasing the bioavailability of iron and iron salts.

Accordingly, in an embodiment of the first aspect of the present invention, the core further comprises a chelating agent. Preferably, the chelating agent is selected from the group consisting of tartaric, malic, succinic, fumaric, citric, lactic and oxalic acids or a salt thereof, EDTA and sucrose. More preferably, the chelating agent is sucrose. The combination of iron or an iron salt and a chelating agent in the formation of a core containing iron alginate with an outer layer containing calcium alginate of the present invention improves the bioavailability of iron and at the same time can isolate it from the environment outside the gastrointestinal tract, which avoids the decay of the matrix containing iron, and the appearance of an unpleasant aftertaste associated with iron salts used to enrich products. As mentioned above, iron sugar is included among the water-soluble iron salts used in the present invention, although, strictly speaking, it is a complex of iron hydroxide and sucrose. The use of iron sugar as an iron salt includes the advantages of a chelating agent due to the presence of sucrose.

The specified iron source product is obtained by the following method, comprising the steps of: (i) forming a core containing iron alginate by contacting at least one bioavailable water-soluble iron salt and at least one water-soluble alginate salt, (ii) contacting the core and an aqueous solution of calcium salt in a concentration of from 0.025 M to a concentration below the saturation point of the solution, and (iii) the allocation of the resulting solid particles. Preferably, at least one bioavailable iron salt used to form the core is iron sugar. In addition, preferably, at least one alginate salt used to form the core is sodium alginate.

The core used to produce the particles can be formed by depositing at least one water-soluble alginate salt on the surface of the particles from at least one iron salt. You can use any method that makes possible the deposition of alginate films on particles of iron salts. Preferably, this is done by spraying an alginate salt solution through a spray nozzle onto particles of an iron salt, which occurs with stirring in conventional solid mixing equipment. This method is intended for equipment such as inclined plates or rotating drums, which may or may not be equipped with auxiliary paddle mixers for the fluidized bed, in which the temperature is maintained and in which the particles continue to move up and down the air flow penetrating the particle layer.

An iron source product is preferably obtained by (i) forming a core by dissolving or suspending at least one bioavailable iron salt in an aqueous solution of at least one water-soluble alginate salt to obtain a gel, (ii) slowly adding the resulting gel to an aqueous solution of calcium salt in a concentration constituting from 0.025 M to a concentration below the saturation point of the solution, with vigorous stirring, and (iii) filtering and washing with water the solid particles obtained.

When the inner core containing iron alginate is formed by dissolving or suspending at least one iron salt in an aqueous solution of at least one alginate salt, crosslinking of the alginate with iron cations occurs throughout the core, making the core itself less soluble in water and having a greater mechanical strength. For the formation of the nucleus, a concentration of sodium alginate above 0.6% wt./mass. (or equivalent concentration when using another water-soluble alginate salt). Thus, preferably, at least one alginate salt is sodium alginate and its concentration in the aqueous solution is at least 0.6% w / w. Lower concentrations lead to the formation of viscous solutions, but not solid particles. The upper limit of the concentration of alginate salt used is determined by its solubility in water and the viscosity of the resulting alginate solution.

The concentration of the iron salt used in the production of the core can be selected as desired. Lower iron concentrations will result in the formation of iron-depleted solid particles, while higher concentrations will lead to the formation of particles with a higher iron content. The upper concentration limit of the iron salt used is determined by its solubility in water. Fully satisfactory results were achieved using both iron and iron salts, in the concentration range up to 1 M. The iron salt is mixed with an aqueous alginate solution with vigorous stirring to avoid too viscous the mixture or precipitate formation.

When subsequently a gel-gel mixture of iron and alginate salt is slowly added to a solution containing calcium salt to form a core with an outer layer containing calcium alginate, solid particles with especially good mechanical strength are obtained. The formation of such particles requires a minimum concentration of calcium above 0.025 M. The maximum concentration is not critical, provided that it corresponds to a concentration below the saturation point of the solution. Any specialist in this field understands well that the saturation point of a solution, that is, the point of maximum concentration, depends on the temperature of the liquid, as well as on the chemical nature of the substances involved.

The concentration of calcium in the cooking process can be used to control the final concentration of calcium in the particles. When adding a core of iron / alginate particles to a calcium solution, grinding equipment can be used to reduce particle size. In the examples below, a laboratory homogenizer was used to convert solid particles into sand paste. Ultimately, the resulting particles are filtered and thoroughly washed with water to remove any free metal cations.

The above product of the source of iron should have a particle size small enough to allow good mixing and not cause their isolation when added to fortified food products, and also in order to have the least organoleptic effect on the final enriched food product. Thus, the preferred particle size of the invention will depend on fortified foods.

The method of obtaining the product of the source of iron used to enrich water-containing food products allows controlling the size of the obtained particles using grinding equipment (the finer the equipment is ground, the finer the particles). In the manufacturing process, macroscopic aggregates of particles can form, for example, aggregates having dimensions of the order of 0.1-1 mm, although smaller formations corresponding to aggregates of fewer particles or to individual particles are also obtained. Thus, it is further possible to classify by particle size by screening the finished product to discard coarse particles. The particle production process allows you to get a very fine powder with barely noticeable particles. Preferably, the average particle size of the iron source product, which may be in the form of particles or some small particle aggregates, is in the range of 5 to 20 microns, although even smaller particles may form.

The particles that make up the product of the iron source, used to enrich water-containing food products, are characterized by excellent load capacity. In addition, they have good stability under standard local conditions of storage and use. Thus, they slightly affect the appearance, taste and maintenance of the quality of fortified foods. In addition, they proved their safety for the human body, that is, they are not toxic when administered orally, while their cytotoxicity is even less than that of the free salt of iron that they contain. As a result of this, these particles are well suited for food fortification, as they are stable for a long period of time in the food matrix and are capable of releasing a water-soluble component of iron upon entry into the gastrointestinal tract.

The amount of iron and calcium contained in the particles can be determined by first solubilizing the particles and analyzing the solution by atomic spectroscopy for quantification. The high iron content of the particles makes it possible to enrich the food to provide the desired absorption of iron by the body even when a small amount of enrichment product is added.

The iron source product used to enrich food products can be added as an iron enriching agent to both dehydrated food products, such as wheat flour and other cereals, as well as food products obtained from them, such as bread, pasta and pastries, and water-containing foods such as yogurt, milk, broths, sauces, juices and other drinks.

The exceptionally high mechanical strength of the solid particles, along with their insolubility in water or in a slightly acidic medium, makes the iron source product particularly suitable for enriching food products that are subjected to aggressive production processes and / or aggressive conditions inherent in the products themselves, such as high water content and acidic environment that occurs in the case of enrichment of yogurt.

The possible incorporation of particulate matter into a food carrier has been tested with yogurt. Yogurt is popular in most populations with a higher propensity for iron deficiency. Therefore, enriching yoghurts will be a great way to combat iron deficiency anemia. The adverse conditions to which particles can be exposed in yoghurts are an indicator of the ability of the particles to protect the environment from the presence of a soluble source of iron. One of the problems with high-fat foods fortified with iron in a high concentration is the oxidation of fat and the development of a rancid taste. This not only reduces the popularity of such a product among consumers, but also reduces its nutritional value.

As indicated in the examples below, enriched yogurt was obtained using the particulate iron source product of the present invention. Iron particles were added to milk, and then pasteurization, homogenization, and fermentation were carried out by adding an enzyme. The results of the tests showed that the particles are able to overcome the difficulties associated with the enrichment of yogurt (both related to the production process and the properties of the carrier itself), since the final product was both visual (no corresponding changes in color or appearance), and organoleptically (no rancid or metallic taste noted) is comparable to unenriched yogurt. In addition, the obtained enriched yogurt containing the iron source product of the present invention showed good storage stability. Therefore, in the case of less problematic media, the task will be further simplified.

Accordingly, in a preferred embodiment, the iron-fortified food product of the invention is yogurt.

In another preferred embodiment, the iron-fortified food product of the invention is milk.

In another preferred embodiment, the iron-fortified food product of the invention is a beverage.

In another preferred embodiment, the iron-fortified food product of the invention is a meat emulsion.

In a further preferred embodiment, the iron-fortified food product of the invention is a sausage.

In addition, the present invention covers all possible combinations of specific and preferred groups described above in this document.

The ranges given, for example, for temperature, time, size, and the like, should be considered approximate unless expressly stated otherwise.

Brief Description of the Drawings

Figure 1 shows the change in body weight in grams in animals in the control group (B) and in animals receiving particles (C). The X axis represents time in days, and the Y axis represents weight in grams.

Figure 2 shows the change in food intake in grams per day per cell in the control group (B) and in the group receiving particles (C).

Figure 3 presents the change in water consumption in grams per day per cell in the control group (B) and in the group receiving particles (C).

Figure 4 shows the percentage iron release (% Fe) under four different storage conditions, where RT = storage at room temperature, 37C = storage at 37 ° C, w = storage in aqueous solution, d = storage without solvent. The X axis represents the time in months.

Figure 5 shows the expressed in percent release of calcium (% Ca) under the four different storage conditions indicated above. The X axis represents the time in months.

Figure 6 shows the ratio of metal cations released into the medium (% Fe /% Ca) after half a month and one month under four different storage conditions mentioned above.

7 schematically depicts the release of metals from a region close to the surface: (A) particles having an inner core of iron alginate and an outer layer containing calcium alginate (represented by the shaded region), and (B) particles in which both metals are homogeneous distributed in an alginate matrix.

FIG. 8 shows periods of breastfeeding, induction of anemia, and recovery from anemia when testing animals in an in vivo bioavailability study.

Figure 9 shows the weight gain during recovery from anemia. Time in days is indicated on the horizontal axis, and weight in grams is indicated on the vertical axis. Groups: M = males, F = females, - = negative control, + = positive control, c = particles.

Additional objectives, advantages and new features of the invention will be set forth in part in the description and in part will become apparent to a person skilled in the art after studying the description, or may be studied in practice of the invention. The following examples and figures are illustrative and are not intended to limit the present invention.

Examples

Example 1

A method of obtaining solid particles with Fe / Ca

In a solution of 1.5 g of sodium alginate in 100 ml of water was dissolved 7.98 g of iron sugar (approximately 35% Fe). Using an extraction funnel, a mixture of sodium alginate / iron sugar was added dropwise in 300 ml of a 0.5M aqueous solution of CaCl ₂ . During the dropwise addition, a suspension of the resulting particles was mixed using a laboratory homogenizer (Diax 900, Heidolph Instruments GmbH). The resulting solids were separated by suction filtration. The particles were suspended three times in distilled water to remove any soluble salt and filtered in vacuo. Received 30 g of wet particles (particles 0).

The same process was carried out using either iron chloride or iron sulfate heptahydrate as an iron salt instead of iron sugar. The following particles were successfully prepared using any of the three different indicated iron salts, as well as various concentrations of the three main components (i.e., iron salts, alginate salts and calcium salts):

Particles 1: iron sugar (35% iron) 1M, CaCl ₂ 0.1M, sodium alginate 1.5%.

Particles 2: iron sugar (35% iron) 1M, CaCl ₂ 0.1M, sodium alginate 3.0%.

Particles 3: iron sugar (35% iron) 1M, CaCl ₂ 1M, sodium alginate 1.5%.

Particles 4: iron sugar (35% iron) 1M, CaCl ₂ 1M, sodium alginate 3.0%.

Particles 5: FeSO ₄ · 7H ₂ O 1M, CaCl ₂ 0.1M, sodium alginate 1.5%.

Particles 6: FeSO ₄ · 7H ₂ O 1M, CaCl ₂ 0.1M, sodium alginate 3.0%.

Particles 7: FeSO ₄ · 7H ₂ O 1M, CaCl ₂ 1M, sodium alginate 1.5%.

Particles 8: FeSO ₄ · 7H ₂ O 1M, CaCl ₂ 1M, sodium alginate 3.0%.

Particles 9: FeSO ₄ · 7H ₂ O 0.1 M, CaCl ₂ 0.1 M, sodium alginate 1.5%.

Particles 10: FeSO ₄ · 7H ₂ O 0.1 M, CaCl ₂ 0.1 M, sodium alginate 3.0%.

Particles 11: FeSO ₄ · 7H ₂ O 0.1M, CaCl ₂ 1M, sodium alginate 1.5%.

Particles 12: FeSO ₄ · 7H ₂ O 0.1 M, CaCl _{2 1} M, sodium alginate 3.0%.

Particles 13: FeCl ₄ 0.1 M, CaCl ₂ 0.1 M, sodium alginate 1.5%.

Particles 14: FeCl ₃ 0.1M, CaCl ₂ 1M, sodium alginate 1.5%.

The resulting particles had a color depending on the iron salt used. The low release of cations from the particles makes them tasteless and suspended in water.

Example 2

Particle Sizes of Iron Source Product

In order to estimate the particle size obtained in Example 1 (particles 1-14), an optical microscope calibrated to measure length was used. In all cases, macroscopic aggregates of particles were clearly visible even to the naked eye, and most clearly when using a microscope. Although these aggregates were of the order of 0.1-1 mm, much smaller formations could be observed, possibly aggregates of fewer particles or individual particles.

A small, barely visible part of the particle sample was placed on a labeled microscope slide. One drop of water was added to the particles. The suspension was slightly mixed with a metal knife and a cover glass was placed on each microscope slide. Each sample was studied using an optical microscope (Nikon Eclipse E800, Nikon Corp., Tokyo, Japan) with a 20 × objective and 10 × eyepiece. For each sample, a region was chosen in which it was possible to observe either individual particles or the smallest clusters of particles. This area was photographed using a digital camera (Soft Imaging Systems, Colorview II) connected to a PC that controls the image analyzer (analySIS3.0, SoftImaging System Corp., Lakewood Co.). A calibrated scale for determining size was superimposed on each image.

The images obtained indicated that the size of the particles and some of the smallest aggregates was usually close to 20 μm, and in some cases decreased to less than 5 μm. Coarser grinding equipment can produce larger particles, although finer particles are generally preferred for food fortification.

Example 3

Particulate Characterization

0.3 g of the wet particles were digested in a microwave in concentrated nitric acid. The concentration of iron and calcium was quantitatively determined by ICP-OES (inductively coupled plasma optical emission spectroscopy).

The amount of iron and calcium in the particles was almost independent of the batch of analyzed particles. Two batches of particles were obtained in accordance with example 1 (particles 0). The concentration of iron and calcium, expressed as a percentage of weight (± standard deviation), in two batches are shown below in table 1.

Table 1 Party 1 Party 2 % Fe 8.6 (± 0.49) 7.93 (± 0.071) % Ca 0.84 (± 0.021) 1.23 (± 0.0074)

For both parties, not only very similar results were obtained, but also an excellent particle loading capacity was observed.

Example 4

Particle cytotoxicity

An in vitro test was performed to verify the absence of toxicity of the particles of the invention containing the iron salt. The test included the incubation of HELA cell line cells with substances whose cytotoxicity was to be tested; this test is widely used as a method of checking toxicity. The tested compounds included particles, as well as their main components separately: the iron salt used in the particles (iron sugar), and sodium alginate. A negative control was also prepared containing only growth medium for HELA, which was used to control growth.

Test media were prepared under sterile conditions. The particles obtained as in example 1 (particles 0; batch 2 of example 3) were sterilized in an autoclave (20 min, 110 ° C). Using sterile material, 72 mg of particles (medium with particles), 17 mg of iron sugar (medium with iron salt) and 18 mg of sodium alginate (medium with alginate) were separately dissolved in 50 ml of growth medium for HELA. The particles were insoluble and remained in suspension. Dilutions of 1: 1, 1:10 and 1: 100 of each of the test media were obtained by diluting the following amounts of media with particles, iron salt or alginate of fresh growth medium for HELA:

1: 1 20 μl + 0 μl fresh medium for HELA

1:10 2 μl + 18 μl fresh medium for HELA

1: 100 0.2 μl + 19.8 μl fresh HELA media

Solutions of 1: 1 particles and iron salts had almost the same concentration of iron (0.285 mg of iron / ml and 0.297 mg of iron / ml, respectively). The same applies to their dilutions of 1:10 and 1: 100.

3,500 HeLa cells were cultured in 36 wells of two 96-well plates: 9 control wells and 27 wells for three repetitions of 3 concentrations of 3 products. Medium up to 100 μl was added to each well. Cells were incubated at 37 ° C for 24 hours. 20 μl of each of the test media and 80 μl of fresh medium were added to each well. Only 100 μl of fresh medium was added to the controls. One of the plates was incubated for 24 hours, and the other for 72 hours. Cell viability was determined after 24 and 72 hours of incubation using the EZ4U non-radioactive test for cell proliferation and cytotoxicity (Biomedica Medizinproducte GmbH).

The results were expressed as a percentage of growth relative to the control, that is, as a percentage of viability, which was calculated as the number of living cells in each of the tested solutions (particles, iron sugar or sodium alginate), divided by the number of living cells in the negative control, and expressed in percentages. The results shown in table 2 below indicate that the cytotoxicity of encapsulated iron is less than that of free iron. Taking into account that iron sugar is a safe food supplement, the encapsulated form is also such. No toxicity was also observed for sodium alginate.

table 2 % viability 24 hours 1: 1 1:10 1: 100 Particles 82.5% ± 2.8 98.7% ± 12.4 100.5% ± 7.5 Iron salt 76.3% ± 9.7 101.7% ± 8.5 95.7% ± 3.2 Sodium Alginate 87.9% ± 6.6 85.8% ± 6.6 79.9% ± 1.7 % viability 72 hours Particles 65.7% ± 8.8 85.2% ± 12.7 80.9% ± 18.6 Iron salt 66.8 ± 23.0 62.0% ± 15.7 68.7% ± 15.3 Sodium Alginate 74.2% ± 6.7 91.7% ± 16.4 80.2% ± 14.7

Example 5

Acute particle toxicity

An in vivo test was performed to assess the absence of toxicity in rats at a dose 100 times greater than that provided for humans. The reference dose for humans was considered two-thirds of the RDI (recommended daily dose) for a person weighing 70 kg (0.14 mg Fe / kg body weight). As a result, a dose of 14 mg Fe / kg body weight was used in this acute toxicity study in rats.

The iron particles used in this test were made as in example 1 (particles 0; batch 2 of example 3). Since the particles were intended for animal consumption, they were made under sterile conditions to avoid contamination of the particles with pathogenic microorganisms.

The animals used are described below:

- Species and line: rats, Sprague-Dawley (SD), Crl: CD, provided by Charles River Laboratories, France.

- Number and type of animals: 12 nulliparous and not pregnant female rats.

- Age (during use of the product): 2 weeks.

- Weight (during product application): 172-193 g.

- Inclusion criteria: ± 20% of average weight at the start of the study.

- Groups: Animals were divided into two groups (control and test), based on the uniform distribution of animal weight in groups.

The study schedule was as follows:

- Day-5: Animals arrive at the facility. Start quarantine in the quarantine room.

- Day-3: End of quarantine. The period of acclimatization in a certain room begins.

- Day 0: Introduction of control or test solutions. The start of the study.

- Day 14: End of study. Pentobarbital euthanasia and necropsy.

Rats spent two days in a quarantine room and the next three days in a specific room for acclimatization. Standard containment conditions were: 20-24 ° C, 30-70% relative humidity (RH), and more than 15 air changes per hour. Temperature and humidity were constantly monitored. A light cycle of 12 hours of fluorescent light and 12 hours of darkness was used. Animals (two in a cage) were provided with food and water (decalcified tap water, filtered and irradiated with UV light) without restrictions.

The introduction of particles and control, as well as observations were performed as described below:

Injected volume: 2 ml / kg of body weight was administered once (day 0 of the study). The control group received only the transport medium (distilled water), and the test group received 87 mg / ml of a suspension of particles (14 mg / kg of body weight of iron).

Interval of administration: The time elapsed between the start of administration to the first animal and the end of administration to the last animal was 45 minutes.

Mortality and morbidity: Both indicators were checked during day 0 at 5, 15, 30, 90 minutes, 2, 4, 6 and 8 hours after administration, and daily until day 14.

Body weight: Registered daily from day 3. On day 0, animals were weighed before administration.

Food and Water Consumption: Recorded three times a week (Mondays, Wednesdays and Fridays), starting on day 3.

Clinical symptoms: Checked after 5, 15, 30, 90 minutes, 2, 4, 6 and 8 hours after administration, and daily for the next 13 days.

All procedures used in this study were based on and strictly followed the following legislative acts: European Commission Directive 2003/63 / EC of June 25, 2003, which amends Directive 2001/83 / EC and relates to medical products for humans . Procedures and equipment for keeping animals were in strict accordance with the requirements for the protection of animals used in experiments:

- European Commission Directive 2003/63 / EC

- European Directive 89/609 / EEC

- Spanish Real Decreto 1201/2005

- FELASA Guide

- OECD Document ENV / JM / MONO (2000) 7

The above parameters were checked for each of the rats, and the control and experimental groups were compared using Student's test with alpha <0.05 (if applicable). Among all animals, no spontaneous mortality was observed. None of the animals showed significant clinical signs indicating the need for killing during the 14-day observation period. No significant difference was found either in body weight (see table 3 below, in which the average values were obtained on the basis of the results for 6 animals; Fig. 1), nor in the consumption of food and water (see table 4 below, in which values were obtained based on results for 3 groups of animals (3 cells); FIG. 2 and FIG. 3, respectively) between groups taking the supplement and not taking the supplement. The values in parentheses correspond to the standard deviation. Macroscopic post-mortem observations were similar for groups taking and not taking the supplement.

From the above results it can be concluded that, due to the absence of mortality or any clinical signs related to the product, the test product does not cause acute toxicity and is not toxic when orally administered to eight-week-old female Sprague-Dawley rats in an equivalent dose of iron corresponding to 14 mg / kg body weight .

Example 6

Particle Stability

The ability of the particles to withstand the release of their payload during storage was tested under various conditions similar to those found in various environments in which to place the particles. Particles were divided into four groups, and each group was subjected to different conditions:

1. Storage at room temperature (RT), aqueous solution

2. Storage at room temperature, particulate matter

3. Storage at 37 ° C, aqueous solution

4. Storage at 37 ° C, particulate matter.

Two different types of conditions in terms of water content were chosen to simulate two diametrically opposed external conditions in which particles are most likely to be placed: liquid foods or foods with a high water content, and dry foods or foods with a low content water. Since food or nutritional supplements are usually stored at room or lower temperatures, and since the higher the temperature, the more aggressive the environment, experiments were carried out at room temperature. They also used a higher temperature for extrapolation to longer storage at lower temperatures, as well as for modeling the behavior of particles in more severe conditions than those that they are likely to encounter.

The iron particles used were made as in Example 1 (particles 0; batch 2 of Example 3). Any traces of iron from the plastic or glass material used in the experiments were removed, leaving it submerged overnight in a 10% (v / v) solution of concentrated hydrochloric acid or nitric acid and rinsing 5-6 times with excess mili-Q water . Two samples and a control were prepared for each analysis under sterile conditions by weighing approximately 120 mg of particles (except control) and adding 15 ml of distilled water (except for experiments with "solid particles"). Each sample was kept sealed at room temperature or at 37 ° C for 0, 0.5, or 1 month. After 0, 0.5, or 1 month, 15 ml of distilled water was added to the “particulate” samples. All analyzed samples were then filtered to remove particulate matter, and the released iron and calcium were quantified in the supernatant by ICP-AOS.

The release of iron and calcium from the particles was used as an indicator of particle stability. The less iron is released, the better is the ability of the particles to maintain the payload in the particles and protect the environment from being destroyed by iron, and the iron from being captured by the environment. The release of iron (average of two samples) under four different storage conditions is presented below in table 6 (see also figure 4). The values in parentheses correspond to the standard deviation.

From the data on the release of loaded into the particles of iron given above, it is seen that the stability of the particles is excellent. Under the most severe conditions, only less than 1.4% of the encapsulated iron is released. By comparing the release rate under various conditions, one can see how the presence of water and storage temperature affect the stability of the particles. Among the four groups, the trend is quite obvious. An increase in temperature (37 ° C instead of room temperature, approximately 20-25 ° C) increases the rate of release, as in the case of contact of particles with a large amount of water. From the previous results, you can also notice that the effect of adding water to the particles or their content in a medium with high water activity has a stronger effect on the rate of iron release than increasing the temperature from room temperature to 37 ° C. The good release rates achieved by storing the particles in aqueous solution and at 37 ° C even improved when the temperature dropped to more realistic values. At room temperature, the maximum release rate is only 0.5410% ± 0.0050 after the first month. The performance of the particles becomes close to excellent if they are also stored at room temperature, but under conditions of low water activity: no iron release was detected during the first two weeks (iron release was less than 0.017%) and only 0.037% ± 0.011 payload lost to the environment after a month of storage.

The results were slightly different for the amount of released calcium, as shown below in table 7 (see also figure 5). The values in parentheses correspond to the standard deviation.

The proportion of calcium released into the environment is more than ten times higher than that for iron, indicating that the ability of particles to retain metals inside is much higher for iron than for calcium. The release of calcium means that the particles are likely to wear out over time, but since calcium is more affordable than iron, calcium is the first to be released into the environment. If the particles were made using only iron, then iron would be released into the environment, not calcium.

Example 7

Particle structure

The production of particles using two different metal cations, iron and calcium, was chosen in order to provide the particles with an additional layer of protection compared to that formed only due to the alginate polymer. The order in which metal cations are added during the production of the particles is also specially selected to promote the formation of an inner core containing iron alginate (i.e., rich in iron) and an outer layer containing calcium alginate (i.e., rich in calcium). This ensures that if the particles wear out during storage or processing, calcium-rich areas will be released into the environment, delaying the release of iron from the core containing iron alginate.

As can be seen from the data obtained in the study of particle stability, under all conditions tested, the proportion of released calcium is much larger than the proportion of iron (see tables 6 and 7 and Fig.6). This fact is fully consistent with the proposed particle structure, where the inner layer contains iron alginate and the outer layer contains calcium alginate. The release of metals from particles is due to the chemical solubilization of their components or the physical deterioration of the particles. In both cases, the first release is from an area close to the surface of the particles. Since the particles are much richer in iron than calcium (7.93% Fe versus 1.23% Ca), most of the calcium should be located close to the surface of the particle, while most of the iron should be located away from it, as shown in FIG. .7 (A). If the distribution of both metals in the particles were completely uniform, as shown in Fig. 7 (B), one would expect that the ratio of released iron / calcium would be close to or equal to that of the particles, and the ratio of released% Fe / released% Ca would be close to 1. The observed ratio is more than 23 times smaller than the ratio predicted based on this model. So this confirms the layered structure of particles having an inner region rich in iron and an outer shell rich in calcium.

Example 8

Bioavailability Tests - Solubility

Fe absorption occurs in the gastrointestinal tract. The pH in the stomach changes during the day, depending on the amount of food in it. In general, the environment in the stomach is quite acidic. On an empty stomach, the pH can reach even 1, increasing to about 5 after a full meal.

Since a full analysis of bioavailability in humans is very complex and time-consuming, the bioavailability of the iron source product of the invention was first assessed using solubility in an artificial stomach as a reference.

The bioavailability of iron particles depends on their ability to release the payload. If the particles are so stable that they withstand the environment of the gastrointestinal tract without releasing their contents, the bioavailability of iron will be close to zero, since the particles are too large to be absorbed in the intestine. On the other hand, if particles are destabilized during passage through the gastrointestinal tract, the payload will be released in the form of a soluble iron salt, which will be absorbed in the intestine, providing high bioavailability of encapsulated iron. An in vitro experiment to evaluate the release of payload during acid cleavage was aimed at detecting the release of iron from particles upon cleavage with hydrochloric acid at pH = 2 close to the pH in the stomach.

In a 10 ml Eppendorf tube, 10 mg of particles with iron sugar, obtained as in Example 1 (particles 1) were weighed, and then 9 ml of water was added. The Eppendorf tube was shaken vigorously by hand to facilitate particle resuspension. Most particles were easily resuspended, although some large particle aggregates remained at the bottom of the tube. A drop of suspension was placed on a microscope slide for inspection. Particles, as well as some larger aggregates, were visible at 100 × magnification. (Olympus CH-2, 10 × eyepiece, 10 × lens). 1 ml of 0.1 M HCl in water was added to the Eppendorf tube and shaken vigorously. A second drop of the acid suspension was also placed on a microscope slide and compared with the previous drop. No major differences between the two drops were observed. After 30 minutes of acid cleavage, a drop of particles was placed on a microscope slide. Before being examined under a microscope, a drop of 1M NaOH was brought into contact with the first drop and the mixture was examined under a microscope. Particle dissolution was almost instantaneous as NaOH diffused through the particle suspension.

A small sample with fresh particles was placed on a clean microscope slide. A drop of 1M NaOH was placed on top of the particles. No changes were observed in the structure of the particles and the particles remained stable during the entire observation time (until the drop evaporated, leaving a precipitate of NaOH with particles included).

The release experiment consisted of two stages. In a first step, the particles are suspended in an acidic medium at an approximate pH of 2. This pH is close to the pH in the stomach. Under such conditions, no visible changes in the structure of the particles were observed, as evidenced by two micrographs obtained at 100 × magnification. This lack of change when suspending particles in distilled water or in 0.01 M HCl is not surprising when considering the chemicals expected in these solutions. In distilled water, the particles are stable, forming a precipitate of iron alginate and calcium alginate. The interaction of iron and calcium with alginate is strong due to the presence of carboxylate groups (-COO ^- ) in the alginate polymer. Acidification of the suspension below the pKa of alginic acid (pKa> 4) converts carboxylate groups (-COO ^- ) to carboxyl groups (-COOH), which have a significantly weaker interaction with cations. This is an important event that leads to the release of the payload of the particles. Although the unprotonated form of alginates (-COO ^- ) is relatively soluble in water, the protonated form is insoluble. This explains the absence of visible changes, since the polymer matrix remains insoluble, although chemically different, which can be demonstrated in the following reaction scheme:

By converting alginate to alginic acid, the release of metals is measured indirectly by solubilizing alginic acid in an alkaline medium. If the cations are released, then the rest is soluble iron and calcium chlorides and insoluble alginic acid. Alkalization of the medium, for example, using 1M NaOH, will lead to the dissolution of the precipitate of alginic acid. This rapid dissolution is due to the conversion of alginic acid back to sodium alginate. Iron and calcium are present in too low a concentration to form a precipitate with sodium alginate, which is soluble in the form of alginate.

On the other hand, if the cations are still bound to the alginate matrix, alkalization converts the alginic acid back to alginate, which is insoluble when iron and calcium are bound. Thus, alkalization of particles carried out for undigested particles should leave them unchanged, since the carboxyl groups present in the particles are already in the form of a carboxylate (-COO ^- ) and strongly interact with both cations, iron and calcium. A small portion of the uncleaved particles suspended in 1M NaOH was also photographed under a microscope, the image remained unchanged over time, which was expected to reveal that the particles are stable if they are not cleaved and stored in 1M NaOH.

The indirect release of metals observed by this experiment shows that after entering the stomach acid pH converts insoluble particles into insoluble alginic acid and soluble salts of calcium and iron. Since soluble iron salts have high bioavailability, the expected bioavailability of these particles is also high. As a consequence, these particles are well suited for enrichment of food products, since they are stable for a long period of time in the food matrix and are capable of releasing metals upon entry into the gastrointestinal tract.

Example 9

Bioavailability - in vivo test

This bioavailability study aims to compare the bioavailability of microencapsulated iron with that of iron sulfate. Iron sulfate was chosen as a positive control because it is not only widely used for standard studies of iron bioavailability, but it also has high bioavailability.

The study was designed to evaluate the ability to make up for iron deficiency in anemic rats using three different sources of iron:

- Negative control group: basic diet depleted in iron, without the addition of iron

- Positive control group: basic iron-deficient diet enriched with 10 ppm iron from soluble iron sulfate

- Test group: a basic iron depleted diet enriched with 10 ppm of iron from microparticles of iron (encapsulated iron sugar).

Three diets were based on the recommended AIN-93G diet for rodent experiments without iron supplementation (reportedly containing 2-6 ppm Fe). A basic diet was used as a negative control. A positive control diet was prepared by supplementing the 10 ppm Fe basic diet with 50 mg / kg FeSO ₄ · 7H ₂ O. A test diet was prepared by supplementing the 10 ppm Fe basic diet with 125 mg / kg microencapsulated iron 7.93% w / w . Fe in microparticles in the form of iron sugar). Diet and distilled water were provided without restriction throughout the study.

Used 40 rats of the Sprague Dawley line (20 males, 20 females) from 4 different litters. Rats were weaned at 21 days of age (day 0, start of study) and randomly seated in stainless steel cages. Two animals of the same sex were placed in each cage. Animals were kept under controlled conditions (20-22 ° C, 30-50% RH, light cycle: 08:00 h-20: 00 h).

Anemia was induced in three groups of animals fed a basic iron-free diet during the first 22 days (females) or 23 days (males) of the study, as shown in FIG. After a period of induction of anemia, the diets in the three groups were changed to

- Negative control: 3 cells with males and 3 cells with females (6 + 6 animals). This group continued to receive a basic diet without the addition of iron.

- Positive control: 3 cells with males and 3 cells with females (6 + 6 animals). This group received a basic diet enriched with 10 ppm Fe in the form of iron sulfate.

- Test group: 4 cells with males and 4 cells with females (8 + 8 animals). This group received a basic diet enriched with 10 ppm Fe in the form of microencapsulated iron (encapsulated iron sugar).

Animals were allowed to recover from anemia for 2 weeks (14 days). After this period, they were anesthetized by inhalation of isoflurane (induction dose: 3%, maintenance dose: 1.5-2%) and euthanized by removing blood through an intracardiac puncture. Animals were weighed periodically throughout the experiment. The amount of food consumed was also measured.

The change in body weight during the recovery period is presented in tables 8 and 9 (see also Fig.9).

The results showed a clear difference between the three different groups, with a much larger and similar increase in body weight in the groups using food fortification, while the group using the basic diet lagged significantly behind the other two groups. Statistically, there were no significant differences between the two groups using food fortification during recovery from anemia. On the other hand, body mass indices in either of the two groups using food fortification (positive control and test group) were significantly higher than the negative control body mass.

The same picture is observed when studying the effectiveness of nutrition. Efficiency is defined as:

$$Effe\mathrm{to}t\mathrm{and}\mathrm{at}n\mathrm{about}\mathrm{from}tbP\mathrm{and}t\mathrm{but}n\mathrm{and}\mathrm{I\; am}=\frac{\mathrm{at}\mathrm{at}el\mathrm{and}hen\mathrm{and}em\mathrm{but}\mathrm{from}\mathrm{from}stel\mathrm{but}(g)}{P\mathrm{about}tReblen\mathrm{and}eP\mathrm{and}u\mathrm{and}(g)}$$

and refers to how food is converted into body tissue. The calculated nutritional efficacy values for the three groups during the recovery period from anemia are presented in Table 10. The values in parentheses correspond to the standard deviation.

Table 10
Efficiency of nutrition for males and females during the recovery period from anemia Nutrition efficiency Negative Control x (SD) Positive Control x (SD) Test group x (SD) Males 0.19 (0.03) 0.43 (0.08) 0.36 (0.04) Females 0.19 (0.02) 0.32 (0.04) 0.28 (0.03) x = mean, SD = standard deviation.

Among the groups of males and females, the differences are significant only between the negative control and any of the groups using food additives, but not between the groups using food additives. Thus, it can be concluded that the differences between iron sulfate and iron microparticles are not statistically significant, but they are statistically significant between the negative control and iron microparticles. As a result, the bioavailability of microencapsulated iron should be the same as that of iron sulfate, but without many of the disadvantages of the soluble iron salt.

Example 10

Fortified yogurt

The inclusion of the particulate iron source product of the present invention in yogurt acting as a food carrier was studied. Particle testing by incorporating them into yogurt was chosen for the following reasons:

- Yogurt has an acidic pH that is aggressive enough to disrupt the particles in the form of iron source products already disclosed in the prior art. This will cause the release of particulate matter and spoilage of yogurt.

- Yogurt has a high water content, also an aggressive environment for other already known iron source products. The water content along with its acidity facilitates the solubilization of iron, which makes enrichment with it more difficult.

- Iron accelerates the oxidation of fats, which enhances the rancid taste of food. In yogurt, about 3% of its content may be fat, which means a large amount of substrate for oxidation if it is in direct contact with the iron component in the enrichment agent.

Enriched yogurt was prepared using particles obtained as in example 1 (particles 0; batch 1 of example 3). A second batch of yogurt was prepared without the addition of iron particles and was used as a control. Yogurt was prepared in the following way:

1. Conditioning and standardization of 25 liters of milk by adding powdered milk with stirring until it is completely dissolved, which ultimately corresponded to 2.5-3% fat.

2. Adding and dispersing 50 g of iron particles with continuous stirring (except for control yogurt).

3. Pasteurization for 90 seconds at 95 ° C.

4. Homogenization at 180 kg / cm ² .

5. The introduction of the enzyme at 45 ° C.

6. Fermentation at 44 ° C for 4 hours until the pH of the yogurt reaches a value of 4.7-4.8.

7. Preservation of yoghurt at 4.5 ° C for up to 1.5 months.

One of the goals of enriched yogurt production was to evaluate the absence of a metallic taste and visually compare the enriched product with the control yogurt. A yogurt tasting was conducted by a group of five unprepared men, none of whom were able to detect even a slight metallic taste in enriched yogurt, although analysis of yoghurts revealed a content of 42.5 ± 4.1 ppm of iron in them. Enriched and control yoghurts were also visually compared, and only a slightly darker color of enriched yoghurt could be detected. Slight staining could occur due to the use of a very dark iron salt, and could only be noticed if yoghurts were placed side by side.

Example 11

Particle Stability in Yogurt

Yoghurts produced with the particulate iron source product of the present invention and control yogurts (without the addition of iron particles) were tested for lipid oxidation, making storage conditions more stringent than usual. Yoghurts were analyzed at a period of 150% of their shelf life, half a month later than their specified shelf life of one month. After this prolonged storage, rancidity was evaluated by gas chromatography. Hexanal is a commonly used marker of rancidity, as it is one of the products of lipid oxidation. A person's ability to detect traces of hexanal is quite high, he is able to feel rancidity if the level of hexanal is higher by about 10 ppm (parts per million, mg / kg of carriers). To achieve the goal that no rancidity was felt or that the hexanal levels were well below the human detection threshold, GC / MS (gas chromatography / mass spectrometry) was used to analyze yoghurts. This method is capable of quantitatively determining traces of hexanal up to 50 ppb (parts per billion, ng / kg), almost three orders of magnitude lower than the detection threshold in humans.

Yoghurts were prepared as in Example 10. Then, in search of traces of hexanal, the yoghurts were analyzed by the GC / MS method.

Both samples analyzed had hexanal levels below 50 ppb, well below the rancidity threshold, which is close to 10 ppm (ppm, ng / g). Thus, particles are a good way to isolate fat in yogurt from a soluble source of iron and avoid oxidation.

Example 12

Fortified meat product and stability

The possible incorporation of microparticles into the food carrier was tested using turkey meat sausages and a meat emulsion such as a Frankfurt sausage. The presence of iron in these samples can lead to fat oxidation if iron is released from the microparticles.

To prepare the Frankfurt sausages fortified with iron, the following list of ingredients and relative amounts were used: pork fillet (40%); bacon (20%); a mixture of additives, spices and flavorings (paymfurt ST-1800, Carinsa Group; 3.4%); vegetable protein (paymprotein ST-91, Carinsa Group; 3%); salt (1.5%); water / ice (27.3%); smoke aroma (0.2%) and potato starch (5%).

Iron microparticles (1 g / final kg) were added together with paymfurt ST-1800.

The method of preparation of Frankfurt sausages was as follows:

1. Chop all the ingredients, chop the meat to a homogeneous state. Use ice to prevent meat from heating.

2. Fill emulsion containers (plastic bags).

3. Boil in a boiler at 75 ° C for 45 minutes.

4. Pack in vacuum and store at 4 ° C.

The following list of ingredients and relative amounts were used to prepare iron-fortified sausages from turkey meat: turkey breast (69%); water (24%); a mixture of additives and flavorings (Carinsa formula CMA-1251 # 1; 6%); corn starch (1%). Microparticles of iron (1 g / final kg) were added together with Carinsa formula CMA-1251 # 1.

For the preparation of sausages used the following method:

1. Prepare the brine by mixing water with corn starch and Carinsa formula CMA-1251 # 1.

2. Chop the meat.

3. Mix the minced meat with brine in a vacuum and soak it for 24 hours.

4. Prepare a homogeneous mass, chopping part of the meat until a homogeneous mass is formed.

5. Fill meat containers (plastic bags).

6. Boil in a boiler at 75 ° C until the internal temperature reaches 72 ° C.

7. Pack in vacuum and store at 4 ° C.

The amount of iron in the samples was measured by ICP-OES (inductively coupled plasma optical emission spectroscopy) after the samples were digested in concentrated HNO ₃ in a microwave oven. The quantification results, expressed in ppm (parts per million, mg / kg), are presented in table 11. The values in parentheses correspond to the standard deviation in percent.

Table 11 Sample No iron added ppm Fe Iron enrichment ppm Fe Frankfurt sausage 12.9 (2.5%) 145 (3.3%) Turkey meat sausage 6.8 (4.4%) 112 (4.7%)

The ability of microparticles not to participate in fat oxidation was tested by counting the amount of hexanal present in the samples. Hexanal is a commonly used marker of fat oxidation, as it is one of the main products of fat oxidation. Its concentration was determined at the beginning and at the end of the shelf life of sausages (day 0 and day 60), and comparisons were made between samples enriched in iron (> 100 ppm Fe) and samples without the addition of iron (~ 10 ppm Fe). Samples were stored in the refrigerator for their shelf life and frozen at -80 ° C until analysis. Hexanal was quantitatively determined by the HS-GC-MS method (gas chromatography in the free space above the product — mass spectrometry). The results, expressed in ppm of hexanal (mg / kg), are presented in table 12. The values in parentheses correspond to the standard deviation in percent.

Table 12 Sausage Day No iron added ppm hexanal Iron enrichment ppm hexanal Frankfurt day 0 0.21 (0.07) 0.35 (0.12) Frankfurt day 60 <0.20 0.22 (0.07) Turkey meat day 0 0.45 (0.16) 0.69 (0.24) Turkey meat day 60 0.55 (0.19) 0.96 (0.33)

A comparison of these results on day 0 shows that although the amount of hexanal may appear to be higher in iron samples, statistically the amount of hexanal cannot be considered as different. Therefore, microparticles effectively prevent the oxidation of fat in the presence of iron during the most aggressive stages of sausage production, namely, their cooking. The same applies to end-of-life results after storing samples for 2 months. The difference in results between the samples enriched in iron and those in which iron was not added is not statistically significant. Moreover, if the same samples were compared at the beginning and at the end of the expiration date, the difference was also not significant. Thus, we can conclude that the presence of iron in the form of iron microparticles does not significantly affect the rate of fat oxidation in the studied meat samples.

Example 13

Resistance of microparticles to homogenization

One of the key stages in the production of many food products, in particular dairy products, is the homogenization stage, in which a liquid or suspension is subjected to high shear stresses. As a result, particles in the suspension, such as fat globules, break down, making the suspension more homogeneous. This shear stress can also cause the destruction of microparticles, the release of their contents (iron) into the supernatant, and, as a result, render protection useless.

To test the extent to which the homogenization of the microparticles releases their contents into the supernatant, 7.9 L of suspension with 10.38 ppm of microencapsulated Fe was prepared using 7.9 L of deionized water and 1.025 g of microparticles. The sample was thoroughly mixed and continuously fed into a single-stage homogenizer, set at a pressure of 20 MPa to 100 MPa. 100 ml of the sample was collected after adjusting the pressure to the desired value and after stabilizing the system. The selected pressure values were: 20 MPa, 35 MPa, 50 MPa and 100 MPa. After homogenization, each sample was left for 8 days without stirring in a closed tank. Immediately before analysis, the samples were microfiltered (0.45 μm filter). Concentrated HNO _{3 was} added to each microfiltered sample to a final concentration of 0.5% (v / v) to stabilize the iron in suspension. Iron in the acidified supernatant was quantified by ICP-OES.

The amount of iron in the supernatant and the corresponding amount of iron released from the microparticles at various homogenization pressures are shown in Table 13 below. The sample designated as homogenized at 0 MPa was not homogenized, and the sample designated as 20 (control sample) was homogenized. at 20 MPa, but contained only deionized water.

Table 13 Homogenization (MPa) Fe (ppm) % released Fe 0 0.15 1.44% twenty 0.16 1.54% 35 0.18 1.73% fifty 0.18 1.73% one hundred 0.19 1.83% 20 (control sample) 0.013 0.125%

As can be seen from the previous results, the stability of microparticles during homogenization is excellent. Even when using high pressures, only a very small fraction of the total amount of iron is released from the microparticles.

Example 14

Production and stability of fortified milk

The incorporation of the particulate iron source product of the present invention into milk as a food carrier was tested. Particle testing by incorporating them into milk was selected for the following reasons:

- Milk is a liquid food product, which facilitates the release in the case of other already known products of the source of iron. This makes fortifying whole milk a more difficult task, unlike skim milk, since the presence of iron leads to the oxidation of the fats present in the milk, leaving a rancid taste.

- Being a liquid, milk cannot easily be fortified with solid foods, as they will tend to settle on the bottom of the container.

Whole cow milk provided by a local farmer was stored in a refrigerator (4 ° C before processing). 35 g of wet microparticles (75% moisture) were dispersed in 600 ml of distilled water. 50 l of milk was treated without adding iron and 50 l of milk was mixed with 600 ml of a suspension of microparticles. In both types of milk was added 1 g / l E-339. First, for 50 l of milk without adding iron, and then for 50 l of milk with adding microparticles of iron: (a) the milk was gradually heated and when the temperature reached 75 ° C, it was homogenized using a two-stage homogenizer (18 MPa for the first stage, 4 MPa for second); (b) when the milk reached a temperature of 90 ° C, it was left at this temperature for 1 min; (c) milk was processed at an ultrahigh temperature (UHT) for 15 seconds at 135 ° C; and (d) the milk was cooled to room temperature and dispensed into 0.5 L non-sterile glass bottles.

After bottling, both types of milk were tested and no metallic taste could be detected. To prevent microbiological spoilage of milk, it was autoclaved at 121 ° C for 15 min. After cooling, milk was stored at 4 ° C. At the end of the shelf life (1 month), the presence of hexanal in milk was quantified.

At the beginning and at the end of the expiration date, the appearance of milk was visually checked. No trace of sediment was observed, but a slightly darker color of enriched milk could be seen. After milk production, its taste and smell were checked. None of the types of milk showed any metallic or rancid smell or taste. In fact, no difference in taste or smell was found between the fortified iron and the non-fortified milk. The absence of rancid odor or taste is fully consistent with a certain concentration of hexanal in both types of milk at the end of the shelf life: hexanal could not be detected in the samples, therefore, if it was present, then at a concentration below 10 μg / l.

Example 15

Microparticle production using calcium acetate

The high concentration of calcium used to form the microparticles can lead to corrosion if the salt used is calcium chloride. The reason is that chloride anions are very aggressive against steel, the material commonly used in industrial tanks, pipes and mixers. In order to avoid these problems and taking advantage of the fact that chloride anions do not play any role in the production of microparticles, a batch of microparticles was obtained using another calcium salt, in particular calcium acetate.

Unlike calcium chloride, which is excellently soluble in water, calcium acetate is less soluble, so the initial concentration of calcium in 5M cannot be reached. Instead, a lower concentration (1.8M) was used.

The method used to obtain microparticles practically did not differ from that used to obtain particles with calcium chloride:

In a solution of 1.5 g of sodium alginate in 100 ml of water was dissolved 16 g of iron sugar (approximately 35% Fe). Using an extraction funnel, a mixture of sodium alginate / iron sugar was added dropwise in 70 ml of a 1.8M solution of Ca (AcO) ₂ . During the dropwise addition, a suspension of the resulting particles was mixed using a laboratory homogenizer (Diax 900, Heidolph Instruments GmbH). The resulting solids were separated by suction filtration. The particles were suspended three times in distilled water to remove any soluble salt and filtered in vacuo. 35.6 g of wet particles were obtained.

The content of Fe and Ca was measured by the method of example 3. The stability of the particles was determined by measuring the release of Fe and Ca on day 0 by the method of example 6.% humidity and% solids were measured in the following way:

1. Weigh the glass beaker ( _empty weight).

2. Weigh approximately 1 g of microparticles into a glass. Weigh the glass again ( _wet weight).

3. Place the beaker in an oven at 110-120 ° C for two hours.

4. Leave a glass with dry microparticles to cool to room temperature for 30 minutes.

5. Weigh the beaker with microparticles again ( _dry weight).

6. Repeat the drying / cooling / weighing cycle until the weight remains constant.

7. Calculate the moisture content as:

$$\%\mathrm{at}l\mathrm{but}\mathrm{well}n\mathrm{about}\mathrm{from}t\mathrm{and}=\mathrm{one\; hundred}\frac{\mathrm{at}e{\mathrm{from}}_{\mathrm{at}l\mathrm{but}\mathrm{well}ns\mathrm{th}}-\mathrm{at}e{\mathrm{from}}_{\mathrm{from}\mathrm{at}x\mathrm{about}\mathrm{th}}}{\mathrm{at}e{\mathrm{from}}_{\mathrm{at}l\mathrm{but}\mathrm{well}ns\mathrm{th}}-\mathrm{at}e{\mathrm{from}}_{P\mathrm{at}\mathrm{from}t\mathrm{about}\mathrm{th}}}$$

Results

- Fe content: 15.0% Fe (w / w) in wet microparticles, 45.4% Fe (w / w) in dry microparticles

- Ca content: 0.6% Ca (w / w) in wet microparticles, 1.9% Ca (w / w) in dry microparticles

- humidity: 66.9% moisture (33.1% solids)

- release of Fe and Ca: 0.2% of released Fe and 8.2% of released Ca.

Example 16

Heat resistance

Further, the suitability of microparticles for food was tested, checking whether the high temperature used in the preparation or sterilization of food affects their ability to hold the payload (iron) inside the microparticles.

1 g of microparticles was weighed into each of two suspended glass beakers. In a drying cabinet at a temperature of 125 ° C, one of the samples was kept for 30 minutes, and the other remained in a drying cabinet for 180 minutes. Then the particles were cooled to room temperature and each glass was weighed again. 40 mg of dry microparticles were suspended in 100 ml of deionized water and the suspension was manually stirred. 1 ml of each suspension was diluted to 10 ml by adding 9 ml of deionized water. Samples were filtered to remove solid particles from the suspension. Then, 50 μl of concentrated nitric acid was added to each sample to stabilize the iron in suspension, and the samples were subjected to ICP-OES to quantify the released iron and calcium.

The microparticle suspension immediately before filtration had a total concentration of 11 ppm Fe. The supernatant after filtration contained only iron released from the microparticles, which was quantified by the ICP-OES method, obtaining the following results:

30 min, 125 ° C: 0.04 ppm Fe

180 min, 125 ° C: 0.03 ppm Fe.

In both cases, less than 1% of the iron present in the microparticles was released from them by heating the microparticles. As a result, microparticles can be considered stable even after treatments that are much more aggressive than those that foods are usually subjected to (3 hours at 125 ° C).

Claims (11)

1. Iron-fortified food product containing an iron source product, an iron source product obtained by a method comprising the following steps:
(i) the formation of a core containing iron alginate by contacting at least one bioavailable water-soluble salt of iron and at least one water-soluble alginate salt,
(ii) contacting the core with an aqueous solution of calcium salt in a concentration of from 0.025 M to a concentration below the saturation point of the solution, and
(iii) recovering the obtained solid product. 2. The iron-fortified food product according to claim 1, wherein the core is obtained by depositing at least one water-soluble alginate salt on the surface of the particles of at least one iron salt. 3. Iron-fortified food product according to claim 1, in which
(i) a core is formed by dissolving or suspending at least one bioavailable iron salt in an aqueous solution of at least one alginate salt to form a gel,
(ii) the resulting gel is slowly added to an aqueous solution of calcium salt in a concentration of from 0.025 M to a concentration below the saturation point of the solution, with vigorous stirring,
(iii) the resulting solids are filtered and washed with water. 4. The iron-fortified food product of claim 3, wherein the at least one bioavailable iron salt is iron sugar. 5. The iron-fortified food product of claim 3, wherein the at least one alginate salt is sodium alginate. 6. The iron-fortified food product of claim 3, wherein the at least one alginate salt is sodium alginate and its concentration in the aqueous solution is at least 0.6% (w / w). 7. An iron-fortified food product containing an iron source product in the form of solid particles, wherein the particles include a core containing iron alginate and an outer layer containing calcium alginate. 8. The iron-fortified food product of claim 7, wherein the core further comprises at least one bioavailable iron salt. 9. Iron-fortified food product according to any one of claims 1 to 8, which is a yogurt, milk, drink, meat emulsion or sausage. 10. The use of an iron-fortified food product as defined in any one of claims 1 to 8 for the prevention and / or treatment of humans to prevent iron deficiency or to reduce iron deficiency in humans. 11. The use of an iron-fortified food product defined in any one of claims 1 to 8 for the enrichment of a food product.

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